Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.     

Correction to: Storage of blue carbon in isolated mangrove forests of the Galapagos’ rocky coast

Wetlands Ecology and Management - In the original publication, the affiliation of second author and acknowledgements were incorrectly published. The correct affiliation and acknowledgements are...     

Evaluation of some cereals, plants and tubers through protein composition

Wild and cultivated maize, sorghum, rice, amaranth, soybean, and cassava were screened for variability in seed storage proteins. Total seed proteins, albumin (Alb-1 and Alb-2), globulin, alcohol-soluble (A1 and A2), and glutelin (G1 and G2) fractions were investigated by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The comparison was done by the obtained protein patterns and their relative amounts. Using quantitative analysis of the protein composition and the electrophoretic patterns, the relationships between total proteins and amount of individual proteins were determined. Electrophoretic patterns of extracted proteins from investigated samples showed that the main protein subunits were concentrated between 10 and 45 kDa. Variation was found in major fractions and minor bands. Electrophoretic patterns of the protein fractions are directly related to the genetic background of the protein and can be identified and used to certify the genetic makeup of wild, cultivated, or newly derived cereals and plants.     

Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions

The in vitro culture of human hematopoietic cells has recently received considerable attention due to its clinical importance. Most studies of the culture and expansion of hematopoietic cells have been performed in static cultures but only very few reports exist on the use of bioreactors where strict control of environmental variables is maintained. In this work, the design, characterization and application of a fully instrumented minibioreactor for the culture of human hematopoietic cells from umbilical cord blood is presented. The system consists of a stirred- tank reactor where cells are maintained in suspension in an homogeneous environment and without the need of a stromal feeding layer. The minibioreactor was coupled to a data acquisition and control system which continuously monitored pH, dissolved oxygen and redox potential. When operated at 75 rpm with a hanging magnetic bar (impeller-to-tank diameter ratio of 0.57), the dead and mixing times were 120 and 80 s, respectively, and the maximum response rate and volumetric oxygen transfer coefficient were 0.8 mM O2 hr-1, and 1.8 hr-1, respectively. Such characteristics allowed a tight control of pH(until day 11) and dissolved oxygen at predetermined set-points, and up to a 7-fold expansion of hematopoietic progenitors was possible in cultures maintained at 20% dissolved oxygen with respect to air saturation. Growth phase and cell concentration could be inferred on- line through determinations of oxygen uptake rate and culture redox potential. Oxygen uptake rate increased during exponential growth phase to a maximum of 40 μM hr-1. Such an increase closely followed the increase in concentration of hematopoietic progenitors. In contrast, culture redox potential decreased during exponential growth phase and then increased during death phase. The designed system permits not only the maintenance of controlled environmental conditions and on-line identification of fundamental culture parameters, but also the application of control strategies for improving expansion of hematopoietic cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.     

Revisiting the influence of learning in predator functional response,how it can lead to shapes different from type III

Predator/parasitoid functional response is one of the main tools used to study predation behavior, and in assessing the potential of biological control candidates. It is generally accepted that predator learning in prey searching and manipulation can produce the appearance of a type III functional response. Holling proposed that in the presence of alternative prey, at some point the predator would shift the preferred prey, leading to the appearance of a sigmoid function that characterized that functional response. This is supported by the analogy between enzyme kinetics and functional response that Holling used as the basis for developing this theory. However, after several decades, sigmoidal functional responses appear in the absence of alternative prey in most of the biological taxa studied. Here, we propose modeling the effect of learning on the functional response by using the explicit incorporation of learning curves in the parameters of the Holling functional response, the attack rate (a), and the manipulation time (h). We then study how the variation in the parameters of the learning curves causes variations in the shape of the functional response curve. We found that the functional response product of learning can be either type I, II, or III, depending on what parameters act on the organism, and how much it can learn throughout the length of the study. Therefore, the presence of other types of curves should not be automatically associated with the absence of learning. These results are important from an ecological point of view because when type III functional response is associated with learning, it is generally accepted that it can operate as a stabilizing factor in population dynamics. Our results, to the contrary, suggest that depending on how it acts, it may even be destabilizing by generating the appearance of functional responses close to type I.     

Joker: An algorithm to insert patterns into sequences for designing antimicrobial peptides

Innovative alternatives to control bacterial infections are need due to bacterial resistance rise. Antimicrobial peptides (AMPs) have been considered as the new generation of antimicrobial agents. Based on the fact that AMPs are sequence-dependent, a linguistic model for designing AMPs was previously developed, considering AMPs as a formal language with a grammar (patterns or motifs) and a vocabulary (amino acids). Albeit promising, that model has been poorly exploited mainly because thousands of sequences need to be generated, and the outcome has high similarity to already known AMPs. Here we present Joker, an innovative algorithm that improves the application of the linguistic model for rational design of antimicrobial peptides. We modelled the AMPs as a card game, where Joker combines the cards in the hand (patterns) with the cards in the table (sequence templates), generating a few variants. Our algorithm is capable of improving existing AMPs or even creating new AMPs from inactive peptides. A standalone version of Joker is available for download at <http://github.com/williamfp7/Joker> and requires a Linux 32-bit machine.